Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic illustration of US-triggered deep-tissue activation of SPINs to release immunomodulators. b Schematic illustration of sonodynamic activation of SPINs to debulk tumor, enhance tumor immunogenicity, and release immunomodulators in situ as well as synergetic action of IDO inhibition and PD-L1 blocking on enhancing antitumor immunity with alleviated irAEs relative to free-drug administration.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Activation Assay, In Situ, Inhibition, Blocking Assay

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Chemical structures of amphiphilic semiconducting polymeric modulators and schematic illustration of their self-assembly and surface modification to form SPINs. b The molar ratios of each component in different SPINs. c Zeta potentials and hydrodynamic sizes of different SPINs in 1× PBS buffer (pH = 7.4) ( n = 4). d Photographs of erythrocytes after incubation with 1× PBS buffer (negative control), 1% Triton X-100 (positive control), and 1× PBS buffer containing SPINs at the concentration of 100 µg/mL for 2 h, followed by centrifugation. e Hemolysis percentages of erythrocytes after incubation with SPINs at different concentrations for 2 h ( n = 4). f Schematic illustration of US irradiation of SPIN D2 solutions covered with a pork tissue. g ESR spectra of 1 O 2 for SPIN D2 (20 µg/mL) after US irradiation (1.2 W/cm 2 , 3 min) without or with coverage of pork tissues at different thicknesses. h Release profiles of aPD-L1 and NLG919 from SPIN D2 (40 µg/mL) after US irradiation for different time ( n = 4). i PD-L1/PD-1 binding activity assay after treatment with free aPD-L1 or SPIN D2 (40 µg/mL) with or without US irradiation ( n = 4). SPIN D2 – US versus SPIN D2 + US: P < 0.0001. Statistical significance was calculated via a two-tailed Student’s t test. *** P < 0.001. In ( g – i ), the power intensity of US irradiation was 1.2 W/cm 2 (1.0 MHz, 50% duty cycle). Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Modification, Incubation, Negative Control, Positive Control, Concentration Assay, Centrifugation, Irradiation, Binding Assay, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schedule for the establishment of primary and distant tumors, triple systemic injection of SPINs (0.2 mL, 0.6 mg/mL) via tail vein, US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min), and analysis of immune responses. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 6) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). SPIN D2 + US versus drug + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus drug: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) receiving different treatments as indicated. e Schematic illustration of treatment of rechallenged tumor mouse models using SPINs. f Growths of rechallenged tumors in Panc02 tumor-bearing mice after injection of saline or SPIN D2 (0.2 mL, 0.6 mg/mL) with US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min) ( n = 5). Saline versus SPIN D2 + US: P < 0.0001. g The survival curves of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 10). h Flow cytometry analysis of populations of effector memory T cells in the spleen of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 4). Saline versus SPIN D2 + US: P = 0.0059. i Differentially expressed gene numbers in tumor tissues of mice after different treatments. j Relative expression of Carl , Hmgb1-ps1 , Hmgb1-ps2 , Cd80 , Cd86 , Cd40 , Pdcd1 , Cd3e , Cd8a , Ifng , Gzmb , Cxcl1 , Cxcl2 , Cxcl9 , Cxcl10 , Cxcl11 , Ccl4 , Ccl5 , Il1b , Il2 , Il6 , Il7 , Il15 , Ido1 , and Cd274 in tumors of Panc02 tumor-bearing mice after different treatments (the experiment was repeated independently five times with similar results). k Unsupervised hierarchical clustering of relative gene expression in tumors of Panc02 tumor-bearing C57BL/6 mice after different treatments ( n = 5). Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic of sono-immunotherapy of subcutaneous pancreatic mouse tumors covered with 5-cm tissue. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 5) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), SPIN 0 or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). The primary tumors were covered with 5-cm tissue under US irradiation. SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) after different treatments for 60 days. e Schematic of US-mediated deep-tissue sonodynamic therapy of orthotopic pancreatic rabbit tumors. f Radiolabeling stability of 131 I-SPIN 0 after storage in saline or 50% serum at 37 °C for different time ( n = 3). g , h SPECT imaging ( g ) and signal intensity ( h ) of orthotopic pancreatic rabbit tumors after systemic injection of 131 I-SPIN 0 (1.0 mL, 1.5 mg/mL) for different time ( n = 4). The white dotted circle indicated tumors. i Computed tomography (CT) imaging of orthotopic pancreatic rabbit tumors after systemic injection of saline or SPIN 0 (1.0 mL, 1.5 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 30 min). The white dotted circle indicated tumors. j Tumor volume of orthotopic pancreatic rabbit tumors ( n = 3) after treatments as indicated for different days. Saline + US versus SPIN 0 + US: P = 0.0108. k H&E staining images of orthotopic pancreatic rabbit tumors after different treatments. The experiment was repeated independently three times with similar results. l Survival curves of orthotopic pancreatic tumor-bearing rabbits ( n = 4) after different treatments for 20 days. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; * P < 0.05, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Radioactivity, Single Photon Emission Computed Tomography, Imaging, Computed Tomography, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a , b Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( a ), and CD3 + CD8 + CTLs ( b ) in blood of mice ( n = 4) at 30 day after systemic administrations of saline, SPIN 0 , SPIN D2 (0.2 mL, 1.2 mg/mL) or free-drug mixture (8 mg/kg body weight for NLG919 and aPD-L1) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). Saline – US versus drug − US: P = 0.0023; saline − US versus drug + US: P = 0.0006; drug + US versus SPIN D2 + US: P = 0.0071 for CD3 + CD4 + Th cells ( a ); saline − US versus drug − US: P = 0.0004; saline − US versus drug + US: P = 0.0001; drug + US versus SPIN D2 + US: P = 0.0093 for CD3 + CD8 + CTLs ( b ). c , d Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( c ), and CD3 + CD8 + CTLs ( d ) in spleen of mice ( n = 4) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0008; saline − US versus drug + US: P = 0.0005; drug + US versus SPIN D2 + US: P = 0.0015 for CD3 + CD4 + Th cells ( c ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P = 0.0002; drug + US versus SPIN D2 + US: P = 0.0049 for CD3 + CD8 + CTLs ( d ). e Representative H&E staining images of liver after 30 days of treatments in different groups (white arrows indicate the infiltrated lymphocytes). The experiments were repeated independently three times with similar results. f Heatmap to show relative fold of cytokine levels in serum of mice after different treatments for 30 days relative to those in saline control group. g , h Serum levels of ALT ( g ) and AST ( h ) in mice ( n = 5) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0010; saline − US versus drug + US: P = 0.0020; drug + US versus SPIN D2 + US: P = 0.0054 for ALT ( g ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P < 0.0001; drug + US versus SPIN D2 + US: P = 0.0013 for AST ( h ). i Summary comparison of the antitumor immunity and irAEs between SPIN D2 -mediated sono-immunotherapy and free-drug treatment. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Flow Cytometry, Irradiation, Staining, Two Tailed Test

Journal: Marine Drugs

Article Title: Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract

doi: 10.3390/md17070421

Figure Lengend Snippet: Simplified image of T-cell activation and cancer cell lysis. Resting T-cells are activated by the interaction of TCR:MHC and CD28:CD80 and CD86, leading to the expression of CTLA4. CTLA4 preferentially reacts with CD80 and CD86 causing activated T-cell lysis. Although imbalanced interaction of programmed cell death-1 (PD-1) on activated T-cell and programmed cell death-ligand 1 (PD-L1) on antigen-presenting cell (APC) causes T-cell lysis, such reaction between activated T-cells and cancer cells expressing PD-L1 will lead to the survival of cancer cells and facilitate cancer cell growth. Suppression of PD-L1 by low molecular weight fucoidan extract (LMF) leads to cancer cell lysis. Adapted from [ , ].

Article Snippet: Inhibitory activity of LMF on PD-1 and PD-L1 binding was examined using PD-1:PD-L1(Biotinylated) Inhibitor Screening Colorimetric Assay Kit (BPS Bioscience, San Diego, CA, USA) following the manufacturer’s protocol.

Techniques: Activation Assay, Lysis, Expressing, Molecular Weight

Journal: Marine Drugs

Article Title: Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract

doi: 10.3390/md17070421

Figure Lengend Snippet: Comparison of programmed cell death-ligand 1 (PD-L1) expression levels in seven cell lines by immunofluorescence staining. Seven cell lines with indicated cell densities were prepared (HT1080 and A549 cells at 2 × 10 4 cells/mL, MCF-7, PC-9, NIH:OVCAR-3, and PANC-1 cells at 3 × 10 4 cells/mL, and TIG-1 cells at 4 × 10 4 cells/mL) and seeded at 100 µL per well of a 96-well black plate followed by 24 h culture. PD-L1 detection was carried out using the anti-PD-L1 antibody, as described in the Methods section. Cells were stained with Hoechst 33342 for 30 min and analyzed using ( A ) IN Cell Analyzer 1000. The fluorescence intensities of each cell line were measured and converted to a numerical form for graphical presentation. For each cell line, six viewing fields per well were analyzed. A549 and PC-9 cells were used as low- and high-level controls for PD-L1 expression, respectively. ( B ) Four representative images acquired in ( A ) were shown. A, HT1080; B, A549; C, PANC-1; D, NIH:OVCAR-3.

Article Snippet: Inhibitory activity of LMF on PD-1 and PD-L1 binding was examined using PD-1:PD-L1(Biotinylated) Inhibitor Screening Colorimetric Assay Kit (BPS Bioscience, San Diego, CA, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Marine Drugs

Article Title: Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract

doi: 10.3390/md17070421

Figure Lengend Snippet: Low molecular weight fucoidan extract (LMF) differentially effects transcription of programmed cell death-ligand 1 (PD-L1) and PD-L2 mRNAs in cancer and normal cells. ( A ) HT1080 and ( B ) TIG-1 cells were treated with varying concentrations (0, 1, 10 µg/mL and 0, 10, 100 µg/mL) of LMF respectively, for 48 h. After treatment, RNAs were isolated and subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses. Values obtained from LMF-treated cells were compared with those of untreated cells using Student’s t -test (* p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001, n = 3).

Article Snippet: Inhibitory activity of LMF on PD-1 and PD-L1 binding was examined using PD-1:PD-L1(Biotinylated) Inhibitor Screening Colorimetric Assay Kit (BPS Bioscience, San Diego, CA, USA) following the manufacturer’s protocol.

Techniques: Molecular Weight, Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Marine Drugs

Article Title: Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract

doi: 10.3390/md17070421

Figure Lengend Snippet: Low concentration of low molecular weight fucoidan extract (LMF) suppresses programmed cell death-ligand 1 (PD-L1) protein expression. ( A ) Western blot detection of PD-L1 protein in total protein lysates from HT1080 cells treated with 10 µg/mL of LMF for 5 days by replacing every 2 days with fresh media containing 10 µg/mL of LMF. Control untreated HT1080 cells treated similarly but without LMF. ( B ) Chemiluminescence protein bands from LMF-treated and untreated samples quantitated and bands from LMF-treated cells compared with that of untreated cells using Student’s t -test. (*** p < 0.001, n = 3).

Article Snippet: Inhibitory activity of LMF on PD-1 and PD-L1 binding was examined using PD-1:PD-L1(Biotinylated) Inhibitor Screening Colorimetric Assay Kit (BPS Bioscience, San Diego, CA, USA) following the manufacturer’s protocol.

Techniques: Concentration Assay, Molecular Weight, Expressing, Western Blot

Journal: Marine Drugs

Article Title: Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract

doi: 10.3390/md17070421

Figure Lengend Snippet: Effect of low amount low molecular weight fucoidan extract (LMF) treatment for 5 days on programmed cell death-ligand 1 (PD-L1) protein expression in HT1080 cells. HT1080 were treated with a low concentration of LMF (10 µg/mL) for 5 days and changes in PD-L1 protein levels were measured by flow cytometer. ( A ) PD-L1 protein expressed on the cell surface of HT1080 cells with (red line) and without (blue line) LMF treatment detected using anti-PD-L1 antibody and histograms were generated. ( B ) Mean fluorescence intensity (MFI) per cell was calculated using histogram data. Student’s t -test (** p < 0.01, n = 3) compares MFI of LMF-treated cells with that of untreated cells.

Article Snippet: Inhibitory activity of LMF on PD-1 and PD-L1 binding was examined using PD-1:PD-L1(Biotinylated) Inhibitor Screening Colorimetric Assay Kit (BPS Bioscience, San Diego, CA, USA) following the manufacturer’s protocol.

Techniques: Molecular Weight, Expressing, Concentration Assay, Flow Cytometry, Generated, Fluorescence

Journal: Marine Drugs

Article Title: Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract

doi: 10.3390/md17070421

Figure Lengend Snippet: Inhibitory activity of programmed cell death-1 (PD-1): PD-L1 binding by low molecular weight fucoidan extract (LMF). ( A ) We used PD-1 neutralizing antibody as a positive control in dose-dependent inhibition of PD-1 and PD-L1 binding. Statistical significance was by ANOVA with Tukey test. The letters, a, b, and c indicate statistically significant differences between each letter ( p < 0.05). ( B ) LMF was used to observe a dose-dependent inhibition of PD-1 and PD-L1 binding (N.S.: Not Significant).

Article Snippet: Inhibitory activity of LMF on PD-1 and PD-L1 binding was examined using PD-1:PD-L1(Biotinylated) Inhibitor Screening Colorimetric Assay Kit (BPS Bioscience, San Diego, CA, USA) following the manufacturer’s protocol.

Techniques: Activity Assay, Binding Assay, Molecular Weight, Positive Control, Inhibition

Journal: Marine Drugs

Article Title: Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract

doi: 10.3390/md17070421

Figure Lengend Snippet: Summary of the results.

Article Snippet: Inhibitory activity of LMF on PD-1 and PD-L1 binding was examined using PD-1:PD-L1(Biotinylated) Inhibitor Screening Colorimetric Assay Kit (BPS Bioscience, San Diego, CA, USA) following the manufacturer’s protocol.

Techniques: Cell Counting, Growth Assay

Journal: Marine Drugs

Article Title: Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract

doi: 10.3390/md17070421

Figure Lengend Snippet: Primer sequences used for qRT-PCR analyses.

Article Snippet: Inhibitory activity of LMF on PD-1 and PD-L1 binding was examined using PD-1:PD-L1(Biotinylated) Inhibitor Screening Colorimetric Assay Kit (BPS Bioscience, San Diego, CA, USA) following the manufacturer’s protocol.

Techniques: